Background- We were encouraged by the results that a short treatment with the CTLA-4 blocking antibody MDX-010 was associated with decreased viral RNA levels in lymph nodes and an increase in the effector function of both SIV-specific CD4+ and CD8+ T-cells, we pursued this approach and tested the efficacy of protracted CTLA-4 blockade treatment with and without vaccination or vaccination alone as a control. We found that protracted CTLA-4 blockade significantly increased T-cell activation and viral replication in primary SIVmac251 infection, particularly at mucosal sites, and increased IDO expression and activity. Accordingly, protracted treatment with anti-CTLA-4 antibody of RMs chronically infected with SIVmac251 decreased responsiveness to antiretroviral therapy and abrogated the ability of therapeutic T-cell vaccines to decrease viral set point. These data provide the first direct evidence that immune activation drives viral replication and suggest caution in the use of long term therapeutic approaches that increase immune activation in the treatment of HIV infection. Another study showed that antiretroviral therapy treatment and virus suppression is sufficient to reduce death ligand but not death receptors in lymphoid tissue of SIV-infected macaques. We studied mRNA expression of molecules involved in programmed cell death (TRAIL and FasL) and their receptors (DR5 and Fas) in blood and lymph nodes collected longitudinally from SIV-infected RMs before and after antiretroviral therapy. TRAIL, FasL, DR5, and Fas expression were elevated in circulating CD4+ T-cells from SIV-infected RMs and antiretroviral therapy reduced both TRAIL and DR5 expression in peripheral blood, but only TRAIL and not DR5, in lymph nodes from the same animals. These findings suggest that antiretroviral therapy is ineffective in reducing expression of apoptotic death receptors in lymphoid tissue and that analysis limited to blood leukocytes may not reveal such a defect. Our results highlight the persistence of an underlying immunologic condition that may prevent therapy-induced restoration of CD4+ T-cells in lymphoid tissues. Interferon alfa Blockade- The early stages of infection present formidable obstacles that the virus must overcome to establish infection. Understanding and exploiting these weaknesses may provide a chance to prevent productive infection in an exposed host. The early stages of genital transmission can not be studied in humans, thus, intra-vaginal SIV infection of RMs represents one of the best animal models in which to study these events. We will test the contribution of the early innate IFN response at the mucosal portal of entry to the establishment and rate of dissemination of SIV infection in RMs. We will inhibit the biological activity of IFN-alpha and IFN-beta by treating RMs with a blocking antibody, 64G12, that targets the receptor for all these interferons. Indeed 64G12 inhibits SIV-AT-2 induced IFN-alpha and TNF-alpha production in pDCs of RM. Specifically, we wish to test the effect of 64G12 on 1) The establishment of intravaginal SIV infection in RMs and the rate of SIV dissemination to mucosal and peripheral LT, 2) viral and CD4+ T-cell dynamics and the SIV-specific immune responses, and 3) the levels of mucosal and systemic immune activation, and disease progression. The early stages of infection present formidable obstacles that the virus must overcome to establish infection. Understanding and exploiting these weaknesses may provide a chance to prevent productive infection in an exposed host. CQ treatment- Earlier in vitro studies indicated that CQ can block HIV envelope glycosylation and HIV replication. However, two small clinical trials reported only modest reductions in plasma virus of 0.4-0.6 log copies/ml. More recently, CQ has been shown to block HIV-induced immune activation as well as HIV-activated IFN-alpha production and PDL-1 expression by plasmacytoid dendritic cells (pDCs) in normal human PBMCs. It should also be noted that CQ was recently reported to enhance astrocyte permissiveness to HIV infection, raising the possibility of complicating and detrimental effects of this drug. We propose to study the in vivo effects of CQ administration on SIV infection of RMs. Anti PD-1 treatment- We found that SIV specific T-cells have a proliferative defect that may explain the inability of vaccines to eliminate infected cells. SIV specific CD8+T-cells express high level of PD-1 and cytokines, and have impaired proliferative capacity in acute and chronic SIVmac251 infection. Programmed Death-1 (PD-1) is a critical mediator of virus-specific CD8+ T-cell exhaustion. We examined the expression of PD-1 on SIV-specific CD8+ T-cells and its possible involvement in the regulation of cytokine production, proliferation, and survival of these cells. We found that the majority of SIV-specific CD8+ T-cells expressed a PD-1(high) phenotype, independently of their differentiation status, in all tissues tested. PD-1 expression gradually declined on CD8+ T-cells specific for SIV-derived epitopes that had undergone mutational escape, indicating that antigen-specific TCR stimulation is the primary determinant of PD-1 expression. SIV-specific PD-1(high) CD8+ T-cells produced IFN-gamma, TNF-alpha, and IL-2 under cognate peptide stimulation but had a reduced proliferative capacity when compared to PD-1 (low) SIV-specific CD8+ T-cells. PD-1(high) SIV-specific CD8+ T-cells were highly susceptible to cell death leading to loss of such cells after in vitro stimulation. Thus, PD-1 may negatively affect the maintenance of effective CD8+ T-cell responses to HIV/SIV, and manipulation of the interaction of PD-1 with its ligands could result in restoration of the T-cell responses in SIV infection. IL-21 treatment- We have studied in detail the immunological damage induced by SIV infection at mucosal sites of infected macaques and found that IL-17 producing CD4+T-cells (Th17), a lineage of effector CD4+ T-helpers recently identified, are infected by SIVmac251 in vitro and in vivo, and their frequency significantly decreases at mucosal and systemic sites within a few weeks of infection. In animals that progress to disease, Th1 cells are over-represented compared to Th17 cells whereas, in SIVmac251-infected elite controller RMs, a normal Th17/Th1 balance is maintained. Indeed, regression analysis of the frequency of Th17 cells at mucosal sites and plasma virus level demonstrates a negative correlation, suggesting their importance in HIV/SIV pathogenesis. Because Th17 cells play a central role in innate and adaptive immune responses to extracellular bacteria, our finding may explain the chronic enteropathy in HIV infection. Thus, therapeutic approaches to reconstitute an adequate balance between Th1 and Th17, like IL-21 treatment, may be considered to ameliorate the clinical course of HIV infection.